Electric Field Mediated Fusion of Protoplasts of Medicago sativa L. and Medicago arborea L.

Abstract

Electroffusion was performed with root and cell suspension protoplasts of Medicago sativa L. and mesophyll protoplasts of Medicago arborea L. In order to obtain the highest yield of binary fusion products, two factorial experiments were carried out to establish the optimal values of protoplast density, AC field strength, number, duration, and voltage of DC pulse for each kind of protoplast. The sensitivity to an electric field depended upon the origin and size of the protoplasts. For homospecific fusion cell suspension protoplasts required the lowest levels of AC field (50 V cm-1) and pulse voltage (1400 V cm-1); mesophyll protoplasts the highest pulse (1750 V cm-1), and root protoplasts the highest AC field (250 V cm-1). Electrofusion of mesophyll and root protoplasts, carried out at AC field = 250 V cm-l, DC pulse = 1750 V cm-1, 3 pulses of 15 μsec each, yielded 5 % heterokaryons. Fusion of mesophyll and cell suspension protoplasts, performed at AC field = 150 V cm-1, DC pulse = 1500 V cm-1, 3 pulses of 15 μsec each, gave 9 % heterokaryons. Fusion products were identified on the basis of their pigmentation (half green and half colorless). They underwent the first mitotic division after 3–6 days of culture. Heterokaryons were manually isolated and developed into minicolonies and calli. The hybrid nature of one fast growing callus was confirmed by the electrophoretic analysis of peroxidase isozymes.